Refrozen TokuKit blood samples preserve cytometry data

The Question We Hear from Translational and Clinical Operation Teams

When whole blood is fixed at clinical sites using the TokuKit, can samples be thawed, aliquoted, re-frozen at −80 °C, and later re-analyzed without impacting data quality or results?

We set out to answer this directly, because for translational and clinical teams, the ability to maximize the value of limited clinical samples is critical.

The Study Design

We evaluated refreeze performance using whole blood from a healthy donor under two conditions:

• Unstimulated samples, to assess immune cell population frequencies
• Stimulated samples, to assess functional marker stability

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Stimulations were used to induce expression of functional markers that would not be detectable at baseline. Stimulated samples were prepared using a defined blend of PMA/PTI-activated whole blood spiked with PHA-stimulated PBMCs and Daudi cells, followed by fixation with the TokuKit to “lock in” functional states. This enabled us to directly evaluate whether functional markers (activation, exhaustion, proliferation, cytotoxicity, etc.) induced prior to freezing are retained following refreeze

Samples were tested across three conditions:

• 0 refreeze: TokuKit-fixed control
• 1 refreeze: thawed, aliquoted, and refrozen once
• 2 refreezes: thawed, aliquoted, and refrozen twice

After initial fixation and storage at −80 °C, refreeze cycles were simulated by thawing, washing, counting, aliquoting, and refreezing samples in 10% Dimethyl sulfoxide (DMSO) /Cell Staining Buffer (CSB) at −80 °C. All samples were ultimately thawed and processed in the same run using a 43-marker mass cytometry panel to ensure direct comparability.

Key Findings: Immune cell populations and functional marker frequencies are well preserved 

Across up to two freeze–thaw cycles, immune cell population frequencies were highly stable:

• <0.5% mean absolute difference between refrozen and non-refrozen samples
• 100% of major immune cell populations remained within ±3% of controls
• ≤2.8% maximum observed difference across all refreeze conditions‍

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Functional marker subset frequencies also remained consistent after refreezing:

• <3% mean absolute difference in functional subset frequencies
• 95% of functional subsets remained within ±13% of controls
• Custom panel markers, including IFNγ, CD80, and CD68, stayed within the maximum observed deviation across refreeze cycles

These results demonstrate that both lineage-level and functional biology are preserved.

Why This Matters for Translational and Clinical Programs

For teams running longitudinal analyses, exploratory follow-ups, or multi-phase clinical studies, this workflow enables you to:

• Aliquot and re-bank TokuKit-fixed whole blood without compromising data integrity
• Run analyses over time from a single clinical draw
• Maintain confidence in high-dimensional immune and functional readouts

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Available Now

‍This refreeze-validated workflow is fully supported through Teiko’s multiparameter cytometry CRO services, combining the whole-blood preservation TokuKit with custom or off-the-shelf mass and spectral cytometry panels and analysis in the Teiko Dashboard. For clinical teams seeking flexibility without sacrificing quality, we provide a path to do more with every sample you collect.